A technique for removing tumourigenic pluripotent stem cells using rBC2LCN lectin.

INTRODUCTION: Tumourigenesis attributed to residual undifferentiated cells in a graft is considered to be a significant issue in cell therapy using human pluripotent stem cells. To ensure the safety of regenerative medicine derived from pluripotent stem cells, residual undifferentiated cells must be eliminated in the manufacturing process. We previously described the lectin probe rBC2LCN, which binds harmlessly and specifically to the cell surface of human pluripotent stem cells. We report here a technique using rBC2LCN to remove pluripotent cells from a heterogenous population to reduce the chance of teratoma formation. METHODS: We demonstrate a method for separating residual tumourigenic cells using rBC2LCN-bound magnetic beads. This technology is a novel use of their previous discovery that rBC2LCN is a lectin that selectively binds to pluripotent cells. We optimize and validate a method to remove hPSCs from a mixture with human fibroblasts using rBC2LCN-conjugated magnetic beads. RESULTS: Cells with the potential to form teratoma could be effectively eliminated from a heterogeneous cell population with biotin-labelled rBC2LCN and streptavidin-bound magnetic beads. The efficiency was measured by FACS, ddPCR, and animal transplantation, suggesting that magnetic cell separation using rBC2LCN is quite efficient for eliminating hPSCs from mixed cell populations. CONCLUSIONS: The removal of residual tumourigenic cells based on rBC2LCN could be a practical option for laboratory use and industrialisation of regenerative medicine using human pluripotent stem cells.

Controlled release of basic fibroblast growth factor from a water-floatable polyethylene nonwoven fabric sheet for maintenance culture of iPSCs.

Basic fibroblast growth factor (bFGF) is an essential supplement for culture media to support the proliferation of human pluripotent stem cells, while preserving their pluripotency. However, it is extremely unstable under cell culture conditions at 37 °C. Therefore, a culture medium supplemented with bFGF needs to be changed every day to maintain an effective concentration of bFGF. This study aimed to create a bFGF-releasing material via simple bFGF adsorption following oxygen plasma treatment by using a water-floatable polyethylene (PE) nonwoven fabric sheet as a bFGF-adsorbent material. Preliminary oxygen plasma treatment enhanced bFGF adsorption onto the sheet by increasing its surface water wettability. Based on the bFGF concentration in the adsorption solution, the resulting bFGF-adsorbed sheet showed different bFGF-release profiles in the culture medium. The bFGF-adsorbed sheet prepared under optimum conditions released bFGF in a sustained manner, maintaining the bFGF concentration in the culture medium of human induced pluripotent stem cells (iPSCs) at ≥10 ng mL-1 even without medium change for as long as 3 d. The bFGF released from the sheet retained its biological activity to support colony formation of iPSCs while preserving their pluripotency. This type of bFGF-releasing sheet can be used as a new form of bFGF supplement for the culture media of stem cells and would make a significant contribution to stem cell-based research and development.

Tracking inorganic foulants irreversibly accumulated on low-pressure membranes for treating surface water.

While low-pressure membrane filtration processes (i.e., microfiltration and ultrafiltration) can offer precise filtration than sand filtration, they pose the problem of reduced efficiency due to membrane fouling. Although many studies have examined membrane fouling by organic substances, there is still not enough data available concerning membrane fouling by inorganic substances. The present research investigated changes in the amounts of inorganic components deposited on the surface of membrane filters over time using membrane specimens sampled thirteen times at arbitrary time intervals during pilot testing in order to determine the mechanism by which irreversible fouling by inorganic substances progresses. The experiments showed that the inorganic components that primarily contribute to irreversible fouling vary as filtration continues. It was discovered that, in the initial stage of operation, the main membrane-fouling substance was iron, whereas the primary membrane-fouling substances when operation finished were manganese, calcium, and silica. The amount of iron accumulated on the membrane increased up to the thirtieth day of operation, after which it reached a steady state. After the accumulation of iron became static, subsequent accumulation of manganese was observed. The fact that the removal rates of these inorganic components also increased gradually shows that the size of the exclusion pores of the membrane filter narrows as operation continues. Studying particle size distributions of inorganic components contained in source water revealed that while many iron particles are approximately the same size as membrane pores, the fraction of manganese particles slightly smaller than the pores in diameter was large. From these results, it is surmised that iron particles approximately the same size as the pores block them soon after the start of operation, and as the membrane pores narrow with the development of fouling, they become further blocked by manganese particles approximately the same size as the narrowed pores. Calcium and silica are assumed to accumulate on the membrane due to their cross-linking action and/or complex formation with organic substances such as humic compounds. The present research is the first to clearly show that the inorganic components that contribute to membrane fouling differ according to the stage of membrane fouling progression; the information obtained by this research should enable chemical cleaning or operational control in accordance with the stage of membrane fouling progression.

A stable chimeric fibroblast growth factor (FGF) can successfully replace basic FGF in human pluripotent stem cell culture.

Fibroblast growth factors (FGFs) are essential for maintaining self-renewal in human embryonic stem cells and induced pluripotent stem cells. Recombinant basic FGF (bFGF or FGF2) is conventionally used to culture pluripotent stem cells; however, because of the instability of bFGF, repeated addition of fresh bFGF into the culture medium is required in order to maintain its concentration. In this study, we demonstrate that a heat-stable chimeric variant of FGF, termed FGFC, can be successfully used for maintaining human pluripotent stem cells. FGFC is a chimeric protein composed of human FGF1 and FGF2 domains that exhibits higher thermal stability and protease resistance than do both FGF1 and FGF2. Both human embryonic stem cells and induced pluripotent stem cells were maintained in ordinary culture medium containing FGFC instead of FGF2. Comparison of cells grown in FGFC with those grown in conventional FGF2 media showed no significant differences in terms of the expression of pluripotency markers, global gene expression, karyotype, or differentiation potential in the three germ lineages. We therefore propose that FGFC may be an effective alternative to FGF2, for maintenance of human pluripotent stem cells.

A medium hyperglycosylated podocalyxin enables noninvasive and quantitative detection of tumorigenic human pluripotent stem cells.

While human pluripotent stem cells are attractive sources for cell-replacement therapies, a major concern remains regarding their tumorigenic potential. Thus, safety assessment of human pluripotent stem cell-based products in terms of tumorigenicity is critical. Previously we have identified a pluripotent stem cell-specific lectin probe rBC2LCN recognizing hyperglycosylated podocalyxin as a cell surface ligand. Here we demonstrate that hyperglycosylated podocalyxin is secreted from human pluripotent stem cells into cell culture supernatants. We establish a sandwich assay system, named the GlycoStem test, targeting the soluble hyperglycosylated podocalyxin using rBC2LCN. The GlycoStem test is sufficiently sensitive and quantitative to detect residual human pluripotent stem cells. This work provides a proof of concept for the noninvasive and quantitative detection of tumorigenic human pluripotent stem cells using cell culture supernatants. The developed method should increase the safety of human pluripotent stem cell-based cell therapies.

Molecular mechanisms for maintenance of G-rich short tandem repeats capable of adopting G4 DNA structures.

Mammalian genomes contain several types of repetitive sequences. Some of these sequences are implicated in various specific cellular events, including meiotic recombination, chromosomal breaks and transcriptional regulation, and also in several human disorders. In this review, we document the formation of DNA secondary structures by the G-rich repetitive sequences that have been found in several minisatellites, telomeres and in various triplet repeats, and report their effects on in vitro DNA synthesis. d(GGCAG) repeats in the mouse minisatellite Pc-1 were demonstrated to form an intra-molecular folded-back quadruplex structure (also called a G4' structure) by NMR and CD spectrum analyses. d(TTAGGG) telomere repeats and d(CGG) triplet repeats were also shown to form G4' and other unspecified higher order structures, respectively. In vitro DNA synthesis was substantially arrested within the repeats, and this could be responsible for the preferential mutability of the G-rich repetitive sequences. Electrophoretic mobility shift assays using NIH3T3 cell extracts revealed heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and A3, which were tightly and specifically bound to d(GGCAG) and d(TTAGGG) repeats with K(d) values in the order of nM. HnRNP A1 unfolded the G4' structure formed in the d(GGCAG)(n) and d(TTAGGG)(n) repeat regions, and also resolved the higher order structure formed by d(CGG) triplet repeats. Furthermore, DNA synthesis arrest at the secondary structures of d(GGCAG) repeats, telomeres and d(CGG) triplet repeats was efficiently repressed by the addition of hnRNP A1. High expression of hnRNPs may contribute to the maintenance of G-rich repetitive sequences, including telomere repeats, and may also participate in ensuring the stability of the genome in cells with enhanced proliferation. Transcriptional regulation of genes, such as c-myc and insulin, by G4 sequences found in the promoter regions could be an intriguing field of research and help further elucidate the biological functions of the hnRNP family of proteins in human diseases.

Unfolding of higher DNA structures formed by the d(CGG) triplet repeat by UP1 protein.

Fragile X syndrome is caused by expansion of a d(CGG) triplet repeat in the 5'-untranslated region of the first exon of the FMR1 gene resulting in silencing of the gene. The d(CGG) repeat has been reported to form hairpin and quadruplex structures in vitro, and formation of these higher structures could be responsible for its unstable expansion in the syndrome, although molecular mechanisms underlying the repeat expansion still remain elusive. We have previously proved that UP1, a proteolytic product of hnRNP A1, unfolds the intramolecular quadruplex structures of d(GGCAG)5 and d(TTAGGG)4 and abrogates the arrest of DNA synthesis at d(GGG)n sites. Here, we demonstrate that the d(CGG) repeat forms a peculiar DNA structure, which deviates from the canonical B-form structure. In addition, UP1 was demonstrated by CD spectrum analysis to unfold this characteristic higher structure of the d(CGG) repeat and to abrogate the arrest of DNA synthesis at the site. This ability of UP1 suggests that unfolding of unusual DNA structures of a triplet repeat is required for DNA synthesis processes.

Fate of DNA replication fork encountering a single DNA lesion during oriC plasmid DNA replication in vitro.

BACKGROUND: The inhibition of DNA replication fork progression by DNA lesions can lead to cell death or genome instability. However, little is known about how such DNA lesions affect the concurrent synthesis of leading- and lagging-strand DNA catalysed by the protein machinery used in chromosomal replication. Using a system of semi-bidirectional DNA replication of an oriC plasmid that employs purified replicative enzymes and a replication-terminating protein of Escherichia coli, we examined the dynamics of the replication fork when it encounters a single abasic DNA lesion on the template DNA. RESULTS: A DNA lesion located on the lagging strand completely blocked the synthesis of the Okazaki fragment extending toward the lesion site, but did not affect the progression of the replication fork or leading-strand DNA synthesis. In contrast, a DNA lesion on the leading strand stalled the replication fork in conjunction with strongly inhibiting leading-strand synthesis. However, about two-thirds of the replication forks encountering this lesion maintained lagging-strand synthesis for about 1 kb beyond the lesion site, and the velocity with which the replication fork progressed seemed to be significantly reduced. CONCLUSIONS: The blocking DNA lesion affects DNA replication differently depending on which strand, leading or lagging, contains the lesion.

Effect of nonsupplemented low-protein diet on very late stage CRF.

BACKGROUND: There are few reports of the effect of a low-protein diet on very late-stage chronic renal failure (CRF), eg, serum creatinine level greater than 10 mg/dL (884 micromol/L). In this retrospective study, we examined the effects of a very low-protein diet in patients with very late-stage CRF. METHODS: A very low-protein diet (0.25 to 0.54 g/kg body weight/d [0.39 +/- 0.01g/kg body weight/d]) without supplementation of essential amino acids or keto analogues was administered to 76 patients with very late-stage CRF who had serum creatinine levels greater than 10 mg/dL (884 micromol/L). Twenty-one patients with the same serum creatinine level and protein intake of 0.55 to 1.2 g/kg body weight/d (0.68 +/- 0.03 g/kg body weight/d) were observed in lieu of controls. RESULTS: Blood urea nitrogen was significantly suppressed to 43.1 +/- 1.9 g/dL (15.4 +/- 0.7 mmol/L) in the low-protein group compared with 111.2 +/- 7.0 mg/dL (39.7 +/- 2.5 mmol/L; P < 0.001) in the control group. The rate of decline in glomerular filtration rate (creatinine clearance) was 36-fold slower with the low-protein diet (-7.1 +/- 1.0 versus -0.2 +/- 0.4 mL/mon, respectively; P < 0.001). Nutritional state in the low-protein group exceeded that of the control group. Consequently, the renal survival rate improved significantly (P < 0.0001). All patients in the control group were initiated on dialysis treatment within 6 months from a serum creatinine level of 10 mg/dL (884 micromol/L). Conversely, 58% of the low-protein group are still on predialysis treatment. CONCLUSION: A severe low-protein diet is effective not only in preventing deterioration in renal function, but also in maintaining nutritional state despite no supplementation of essential amino acids or keto analogues, even as serum creatinine level is more than 10 mg/dL (884 micromol/L).